Acid and alkaline phosphatase enzyme activity associated respectively with Type I and Type II matrix vesicles play important roles in the mineralization of hard tissues. In vitro and in vivo studies demonstrate that biomechanical loading affects activity of these enzymes in cartilage. Ultrastructural evidence also suggest that both pathological and normal variations in mechanical loads alter matrix vesicle activities in cartilage. Thus, available data suggest that biomechanical loading may modulate mineralization via effects on matrix vesicles and their associated enzymes. The long-term objective of this project is to determine mechanisms by which biomechanical stimuli are transformed into cellular responses in bone and cartilage with particular emphasis on biomechanical regulation of calcification. The specific aim is to investigate one aspect of chondrocyte response to biomechanical signals, i.e. production of matrix vesicles and their associated enzymes. Investigation of the mechanisms by which biomechanical factors regulate calcification could lead to significant progress in prevention and treatment of osteoarthritis, osteoporosis, and skeletal growth disorders. In the proposed project, one group of rats will be fed hard, pelleted rat chow (control diet) and the other group will be fed moistened, ground rat chow (experimental diet) for 4 weeks. The experimental diet requires little chewing and results in reduced biomechanical stimuli to the mandibular condylar cartilage. At sacrifice, the mandibular condyles will be removed, fixed, and decalcified. Frozen thick sections will be incubated in media containing substrates for either alkaline or acid phosphatase. These sections will be post-fixed in osmium, embedded in Epon, and thin-sectioned. Transmission electron micrographs will be taken of matrix vesicles in the proliferative, maturing and hypertrophic zones at 40,OOOx. Number and area of vesicles negative and positive for each enzyme will be measured using a computer image analysis system. Results will be compared in the normal-loading group (control diet) and reduced-loading group (experimental diet) in order to determine the effects of biomechanical factors on matrix vesicle-associated calcification in the growing condyle.